Hereditary non-syndromic hearing loss affects 1:500 newborns and age related hearing loss afflicts the majority of the elderly population. In most forms of hearing loss, hair cell (HC) degeneration is a common final pathway. The long-term goal of our research is to decipher the cell type-specific transcriptional and signaling cascades that are necessary for inner ear development, and to apply this knowledge to identify: (a) genes necessary for hearing; and (b) potent regulators of differentiation, which could ultimately be used to restore hearing. ATOH1 is a master regulator of HC-fate that likely functions through the activation of a series of transcription factors (TFs) as well as non-regulatory genes. However, forced expression of ATOH1 in deafened ears is not sufficient to generate mature auditory HCs. Therefore, there is a critical need to identify the signaling cascades mediating the differentiatio of newly formed HCs. In this proposal we focus on the GFI1 and RFX TFs, whose orthologs in Drosophila are immediate targets of the ATOH1 ortholog. We recently identified an evolutionarily conserved role for the ciliogenic RFX proteins in regulating the newborn mouse HC-specific transcriptome, and identified Rfx3 and Rfx7 as the most abundant Rfx transcripts in HCs. In Specific Aim 1 we will determine the role of RFX in HC differentiation using conditional knockout mice, and identify and validate the RFX-regulated target genes in HCs. In Specific Aim 2 we will identify the molecular targets of GFI1, a gene necessary for outer HC survival and vestibular HC differentiation, and validate the roles of newly identified targets in vivo. In orderto facilitate dissemination of data generated in this and other proposals in the public domain, and as a step towards building the transcriptional networks necessary for inner ear cell type-specific differentiation, we will build and maintain the gEAR - a gene Expression for Auditory Research portal.